The corneal endothelium is a non-regenerative cell monolayer on the internal surface of the cornea, separating the corneal stroma from the anterior chamber fluid (see FIG. 1). The corneal endothelium is responsible for maintenance of corneal clarity by a continual process that prevents excessive hydration of cornea from an influx of cations and water molecules into the collagenous corneal stroma, generally referred to as ‘deturgescence’.
Fuchs Endothelial Corneal Dystrophy (FECD) is a common inherited, corneal endothelial degeneration disorder associated with the presence of corneal guttae, which are microscopic collagenous accumulations under the corneal endothelial layer. After the age of 40, up to 5% of US adults exhibit corneal guttae. The presence of guttae is indicative of FECD but generally represents mild disease that is completely asymptomatic. Advanced (severe) disease develops in a small proportion of patients with guttae. Advanced FECD is characterized by extensive guttae, endothelial cell loss, corneal edema, corneal clouding and consequential vision loss due to corneal edema and clouding. Corneal edema, clouding and subsequent vision loss are a direct consequence of endothelial cell degeneration and loss of deturgescence. Vision loss due to FECD is the most frequent indication requiring full thickness corneal transplantation (penetrating keratoplasty), accounting for greater than 14,000 procedures annually in the US alone. No other treatments are available for FECD. Although corneal transplantation is a largely successful treatment it has the disadvantage that it is invasive and associated with approximate 30% rejection rate, which is not dissimilar to other solid organ allografts. An alternative approach in which just the corneal endothelium is replaced (endokeratoplasty) can also be carried out, but only by very experienced surgeons. Both interventions suffer from lack of donor material, either transplantable corneal buttons or corneal derived endothelial cells derived from donor corneas. FECD is also a risk for other procedures such as cataract surgery and is contraindicated for refractive surgery such as Laser-Assisted in situ Keratomileusis (LAISK) as these techniques lead to additional corneal endothelial cell loss.
FECD segregates into early-onset FECD and age-related FECD, which may be different diseases since guttae are not typically present in early-onset FECD. Early-onset FECD is rare and has been linked to genes such as Col82A2, encoding the α2-subunit of collagen VIII, a component of the endothelial basement membrane. In age-related FECD certain rare autosomal dominant mutations have been found in different genes, such as KCNJ13 (a potassium channel), SLC4A11 (a sodium-borate co-transporter) and ZEB1 (the Zinc-finger E-box homeodomain protein 1). Importantly however, the genetic basis of the majority of autosomal dominant age-related FECD has been attributed to the Transcription factor-4 (TCF4) gene following a genome-wide association study (Baratz K H et al. E2-2 protein and Fuch's corneal dystrophy. N Engl J Med 2010 363:1016-1024). In these studies a Single-Nucleotide Polymorphism (SNP) was identified within an intron of the TCF4 gene: rs613872 on chromosome 18q21.2, which segregated specifically in age-related FECD patients. The increase in the risk of FECD development is calculated as a 30 fold increase in homozygous subjects and the rs613872 marker was able to discriminate between cases and controls with 76% accuracy. At least two regions of the TCF4 locus have been associated with development of FECD, following prior observations of FECD associating with a chromosomal region located at 18q21.2-18q21.32 (Sundin O H et al. A common locus for late onset Fuchs corneal dystrophy maps to 18q21.2-q21.32. Invest Ophthalmol Vis Sci 2006 47:3919-3926). Several other studies illustrated that the presence of a TCF4 trinucleotide repeat (TNR) was more predictive of FECD than the rs613872 marker (Wieben E D et al. A common trinucleotide repeat expansion within the transcription 4 (TCF4, E2-2) gene predicts Fuchs corneal dystrophy. PLoS One 2012 7:e49083; Wieben E D et al. Comprehensive assessment of genetic variants within TCF4 in Fuchs' endothelial corneal dystrophy. Invest Ophthalmol Vis Sci 2014 55:6101-6107; Mootha V V et al. Association and familial segregation of CTG18.1 trinucleotide expansion of TCF4 gene in Fuchs' endothelial corneal dystrophy. Invest Ophthalmol Vis Sci 2014 55:32-42; Stamler J F et al. Confirmation of the association between the TCF4 risk allele and Fuchs endothelial corneal dystrophy in patients from the Midwestern United States. Ophthalmic Genet. 2013 34(1-2):32-4; Kuot A et al. Association of TCF4 and CLU polymorphisms with Fuchs' endothelial dystrophy and implication of CLU and TGFBI proteins in the disease process. Eur J Hum Genet. 2012 20(6):632-8; Thalamuthu A et al. Association of TCF4 gene polymorphisms with Fuchs' corneal dystrophy in the Chinese. Invest Ophthalmol Vis Sci. 2011 52(8):5573-8; Xing C et al. Transethnic replication of association of CTG18.1 repeat expansion of TCF4 gene with Fuchs' corneal dystrophy in Chinese implies common causal variant. Invest Ophthalmol Vis Sci. 2014 55(11):7073-8; Nanda G G et al. Genetic association of TCF4 intronic polymorphisms, CTG18.1 and rs17089887, with Fuchs' endothelial corneal dystrophy in an Indian population. Invest Ophthalmol Vis Sci. 2014 55(11):7674-80).
Unstable repeats are found in a variety of gene regions, such as in the coding region of the gene causing Huntington's disease (HD), whereby the phenotype of the disease is brought about by alteration of protein function and/or protein folding. Unstable repeat units are also found in non-coding regions, such as in the 3′-UTR of the DMPK gene causing Myotonic Dystrophy type 1 (DM1), in the 5′-UTR in the FMR1 gene causing Fragile X syndrome, and in intron sequences such as in the first intron of the ZNF9 gene causing Myotonic Dystrophy type 2 (DM2). DM1 is the most common muscular dystrophy in adults and is an inherited, progressive, degenerative, multi-systemic disorder of predominantly skeletal muscle, heart and brain. DM1 is caused by expansion of an unstable trinucleotide (CTG)n repeat (as noted above, in the 3′-UTR of the DMPK gene). DM2 is caused by a tetranucleotide (or quatronucleotide) (CCTG)n repeat (a quatronucleotide repeat hereinafter being referred to as “QNR”) expansion (as noted above, in intron 1 of the ZNF9 gene). Instability of TNRs is also found to be the predominant cause of several other disorders, such as X-linked Spinal and Bulbar Muscular Atrophy (SBMA), several spinocerebellar ataxias (SCA gene family), C90RF72-associated Amyotrophic Lateral Sclerosis, Frontotemporal Dementia (C90RF72 ALS/FTD), and FECD.
Excessive TNR expansions may lead to a phenomenon referred to as ‘RNA toxicity’, which is the predominant cause of the diseases mentioned above. What happens is that these repetitive elements are transcribed into toxic ‘gain-of-function’ RNAs, which manifest as dominant-negative pharmacology, in which a single disease allele may already cause the disease despite the presence of a normal allele. In the case of DM1, RNA toxicity becomes manifest at the level of mRNA processing when splice regulators, such as the MuscleBlind-Like 1 (MBNL1) protein and CUG-triplet repeat binding protein 1 (CUGBP1) are sequestered from their normal cellular function: the proteins bind to the excess TNRs. Such protein-RNA complexes can be visualized in DM1 cells as nuclear RNA foci (Mankodi et al. Ribonuclear inclusions in skeletal muscle in myotonic dystrophy types 1 and 2. Ann Neurol 2003 54(6):760-8). MBNL1 is a splicing regulator but also binds 3′-UTRs, which therefore also leads to mis-regulation of alternative polyadenylation in DM1 (Batra et al. Loss of MBNL1 leads to disruption of developmentally regulated alternative polyadenylation in RNA-mediated disease. Mol Cell 2014 56(2):311-22). Recent reports suggest that repeat RNAs may be translated into toxic protein species (Cleary and Ranum. Repeat associated non-ATG (RAN) translation: new starts in microsatellite expansion disorders. Curr Opin Genet Dev 2014 26:6-15).
FECD was observed to be associated with a (CTG)n TNR expansion in an intron region of the TCF4 gene that is different from the intron in which the rs613872 marker is located (Mootha et al. 2014; Wieben et al. 2012). It was shown that 79% of FECD patients (noted in leukocyte DNA) had 50 or more repeats (150 nucleotides), whereas 95% of case controls had repeat lengths of less than 40, which shows that a repeat length of 50 or more is highly predictive of FECD, whereas fewer repeats, between 40 and 50, also contribute to appearance of the disease. It is generally accepted in the field that the appearance of a TNR expansion at a size equal or greater than 40 repeats in the TCF4 gene is predictive of disease and indicative of a potential RNA toxicity mechanism leading to FECD (Du et al. RNA toxicity and missplicing in the common eye disease Fuch's endothelial corneal dystrophy. J Biol Chem 2015 290(10):5979-90). RNA foci were identified in fibroblasts from FECD patients that were both homozygous and heterozygous for TNR expansions in the TCF4 gene. No RNA foci were found in fibroblasts from unaffected individuals. Unaffected individuals generally appear to carry wild type TCF4 genes with around 20 TNRs. Heterozygote FECD patients (with fibroblasts wherein RNA foci were detected) carried one normal length allele (20 TNRs) and one allele with an expansion of NM TNRs. In homozygote FECD patients both alleles contained ≥40 TNRs. Consequently fewer than 40 repeats in the TCF4 expanded TNR regions can be considered a non-disease causing genotype. RNA foci were also identified in the corneal endothelium of FECD patient samples, while none were found in unaffected individuals. The presence of such RNA foci appeared associated with a change in the RNA splicing patterns for a number of other genes (Du et al. 2015). These splicing pattern changes are consistent with similar changes noted in DM1 (Wheeler et al. Correction of CIC-1 splicing eliminates chloride channelopathy and myotonia in mouse models of myotonic dystrophy. J Clin Invest 2007 117(12):3952-7; Savkur R S et al. Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy. Nat Genet 2001 29(1):40-7; Li Y J et al. Replication of TCF4 through association and linkage studies in late-onset Fuchs endothelial corneal dystrophy. PLoS One. 2011 6:e18044). The general conclusion is that the majority of FECD cases is caused by RNA toxicity in the corneal endothelial cells due to the presence of TNR expansions in intronic RNA derived from the TCF4 gene. RNA toxicity was found in patients that were either heterozygous or homozygous for the extended repeat, and is likely the result of sequestration of proteins that interact with the RNA harboring the TNR expansions. Such proteins—through this sequestration—can no longer perform their normal function in the cells.
Despite the good results that can be achieved with full corneal transplantation or transplantation of the endothelial layer to treat FECD, it is clear that such procedures still encounter great disadvantages, which have been outlined above. Hence, there remains an unmet medical need to treat patients suffering from, or that are at risk of developing FECD, preferably by means that are a proper alternative for transplantation.